Project 2, Summary/Abstract This is a new project headed by Dr. Daniel DiMaio to determine the mechanism of ?-secretase action in HPV entry. High-risk HPV, most notably HPV16, are an important cause of cancer worldwide. Thus, there is a pressing need to understand the mechanisms of HPV infection and to develop new approaches to inhibit this process. With the support of this grant, we conducted a genome-wide siRNA screen to identify cellular factors required for HPV16 infection and discovered that incoming virus traffics in a retrograde fashion to the Golgi and endoplasmic reticulum. The strongest hit on the screen was a subunit of ?-secretase, a protease which cleaves transmembrane proteins within their membrane-spanning domain to generate biologically active protein fragments. This result is consistent with work from other laboratories showing that ?-secretase inhibitors block HPV infection. Thus, ?-secretase is an important tool to probe the HPV entry pathway, and components of the ?-secretase-dependent entry pathway are potential microbicide targets to prevent HPV infections in parts of the world where the cost of HPV vaccination is prohibitive or in patients at high risk of infection. However, there is no information regarding the mechanism of ?-secretase action during HPV infection. We recently showed that ?-secretase is required for authentic HPV, that this requirement persists in cells that do not express HPV oncogenes (such as hTERT-immortalized foreskin keratinocytes), that ?-secretase inhibition blocks trafficking of incoming HPV between the early endosome and the Golgi apparatus, and that the L2 capsid protein is required for ?-secretase sensitivity. We hypothesize that ?-secretase cleaves cellular proteins to generate products that are required for proper trafficking or to inactivate substrates that inhibit trafficking. To determine the role of ?-secretase in HPV16 infection, we will first identify the ?-secretase-dependent step in HPV infection, utilizing new assays we are developing for various steps in infection. Second, we will determine the role of ?- secretase substrates in infection, focusing initially on Jagged-2 and betacellulin, which play opposing roles in infection. If warranted, we will identify and characterize additional ?-secretase substrates involved in infection and test the possibility that HPV proteins themselves are substrates. Finally, we will test a number of mechanistic hypotheses regarding the role of ?-secretase. For example, we will test whether ?-secretase is required for complex formation between HPV and required cellular factors. In addition to illuminating aspects of HPV infection and possibly suggesting new antiviral approaches, these studies are likely to reveal new features of intracellular trafficking and elucidate novel actions of ?-secretase on the biology of cells.